The invention relates to a novel compound oleanane triterpenoid oligoglycoside of formula 1 of the accompanying drawing (trivially called corniculatonin). The invention also relates to a process for the isolation of the novel compound from a mangrove plant Aegiceras corniculatum (Blanco) belonging to the family Myrsinaceae. It also discloses the antifungal properties of the said compound. The invention further relates to the use of the said compound as a food preservative.
Aspergillus sp. and many other moulds are often common contaminants of food, and effect the quality of the contaminated food by reducing starch and fat contents and lowering digestible energy level (William K. C., Blaney B. J. The potential of mould to reduce the value of foodstuffs. Proceeding of the aquaculture nutrition workshop. Allan G L, Dall W. Eds. Salmadar Bay, NSW-Australia. NSW. Fisher""s 1992, 205-213). Aspergilli are known to occur on cereals, treenuts, peanuts, cotton, fruits, meat, poultry and sea food products (Jay J. M. 1978. Modem food microbiology, sec. Eds. D. Van Norstrand Company, New York.). Aspergillus fresenii and A. niger, used in the present investigation were found to be commonly associated with food products of wheat in coastal regions of India having warm and humid climate. The fungi from the genera Aspergillus are known to produce mycotoxins such as aflatoxins, fumronosins, fusaric acid, TA/AAL toxins, zearalenone, and trichothecene, 5-butyl picolinic acid and related phytotoxic pyridine derivatives. These mycotoxins are highly toxic to a variety of species including plants and humans and can be found in commercially prepared foodstuffs including milk and milk products, beans, cereals, sweet potatoes and commercially prepared animal feeds.
It is evident that new methods and formulations are needed to control plant and animal pathogens and the level of toxic metabolites present in consumable product and in the environment in general. The widespread use of pesticides, fungicides and chemical preservatives, however, has resulted in the development and evolution of resistant pathogens. As environment and health care concerns continue to mount, it will be necessary to identify or develop new fungicides to meet the environmental standards of the future. Particularly those, which are natural products for consumption by animals and thus have, low animal and environmental toxicities.
During the applicant""s search for biologically active molecules from marine organism of Indian Ocean region, crude methanolic extract of a mangrove plant, A. corniculatum exhibited promising central nervous system depressant activity. In order to simplify the procedure for the isolation of the active principle, the crude extract was fractionated. The aqueous fraction being the major fraction, excess was left at room temperature. Surprisingly, it was observed that even on prolonged standing there was no fungal growth on the surface of the aqueous fraction. This indicated that the fraction must contain some molecule acting as antifungal agent. This observation coupled with the facts of increase in antibiotic resistant microbes and paucity of effective antifungals indicated a need for new antimycotic agents. It is with this goal in mind that the applicants studied the a aqueous fraction leading to the isolation of an active saponin.
Saponins are steroid or triterpenoid glycosides widely distributed in plants. The saponins have diverse biological activities finding use in agents employed as fungicides, insecticides, anticancer agents, cosmetics, food preservatives and fertilizers with growth promoting effects. Further use for saponins is in the removal of cholesterol from dairy products and as feed supplements for livestock such as chickens to reduce cholesterol levels in eggs.
A food preservative is containing saponin from aloe woods and p-hydroxy benzoic ester is disclosed in Japanese patent application no. 05009123. An antifungal drug containing saponin extracts from asparagus is described in Japanese patent application no. 2157205. The antimicrobial and pest activity of a saponin isolated from fruit pulp, tobacco and seed are described by Okunji et at. (Int. J. Crude Drug Res., 1990, 28:193-199); Gruenweller et al. (Phytochem., 1990, 29:2485-2490) and by Lalithat et al. (Int. Pest Control, 1988, 30:42-45) respectively. U.S. Pat. No. 4,477,361 (Sperti G. S. et al., 1984) describes a cinnamic compound containing an antimicrobial surfactant which is rendered substantive to the surface being washed. Film forming and/or anti-transpirants coating polymers such as NaHCO3 and light paraffinic petroleum oils have been reported to control the level of fungal colonization (Horst. et al. Plant Disease, 1992, 247-251; Elad et al. Phytoparasitica, 1989, 17:279-288 and Ziv, et al. Hort. Science, 1993,124).
Triterpene sapogenins from the bark of Aegiceras majus (Gaerten) (Syn. Aegiceras corniculatum) have been investigated by Rao and Bose, who initially reported isolation of aegiceradienol (Science and Culture, 1958, 24:486), genin A (Rao K. V. et al., J. Ind. Chem. Soc., 1959, 36:358); aegiceradiol (Rao K. V. et al., Tetrahedron, 1962, 18:461) and norechinocystadienol (Rao K. V. et al., J. Org. Chem., 1962, 27:1470) as products of usual acid hydrolysis of saponin mixture. Aegicerin (Rao K. V., Tetrahedron, 1964, 20:973) is reported to be present in the non-glycosidic extract of the bark of A. majus (Gaerten). Later on Hensens and Lewis (Henses O. D. et al., Tetrahedron letters, 1965, 4639) demonstrated that aegicerdiol or aegiceradienol are artifacts formed during the usual fairly vigorous hydrolysis conditions used for the crude glycosides mixture.
Isorhamnetin, echinocystic acid and rapanone are also known to be constituents of Aegiceras corniculatum (Henses O. D. et al., Aust J. Chem., 1966, 19:169). This mangrove is known to photosynthesize aspartate and alanine as major products of short time photosynthesis (Bhosale L. et al., Photosynthetica, 1983, 17:59). Payne reports the presence of fish toxin, 5-O-methyl embelin in this mangrove (Payne A. M., Diss. Abst. Int. PT. B. Sci. and Eng. 1991, 51:225). Chattopadhyay et al, (Chattopadhyay S. et al., Proceedings of the National Symposium on Marine Resources, Techniques, Evaluation and Management; Vaidyanadhan R. et al., eds., 1990, 5-7) found lupeol, amyrin, oleanolic acid and ursolic acid besides campesterol, stigmasterol and sitosterol, to be the major triterpenoids and steroids respectively. Saponin fraction of Anagallis arvensis is antiviral being effective against Herpes simplex virus type I and polio virus (Amoros M et al., Ann. Pharm. Fr., 1977, 35:371; PI. Med. Phytoh., 1979, 13:122 and Amoros M. et al., Phytochem, 1987, 26:787). Its crude glycoside, Heteroside I, wherein anagalligenone is the constituent genin is reported to be antifungal and to inhibit germination of linseed. Its LD50 in mice was 675 mg/Kg per os and 30.4 mg/Kg by s.c. (Staron T. et al., Phytiat-Phytopharm, 1969, 18:161). The saikosaponins of Bupleurum falcatum, a crude drug in oriental medicine for treatment of hepatobiliary and anti-inflammatory diseases, have shown several metabolic actions in rat, in addition to anti-inflammatory action (Yamamoto M. et al., Arzneim Forsch, 1975, 25:1021,1240).
A comparative study of saikosaponins of Bupleurum longeradiatum var. breviradiatum and B. falcatum showed that per os administration of 1.0-3.0 g/Kg of the saponin was non toxic and the analgesic effect of the former was twice that of later. Both (500 mg/Kg os) prolonged sleeping time produced by hexabarbital sodium in mice (Imaoka, I. et al., Shioku Igaku Zasshi, 1970, 26:1; Chem. Abstr., 1970, 73:43769).
Sakurasaponin from Rapanea melanophloeos (L.) Mez (Myrsinaceae) exhibits molluscidal at 3 ppm and antifungal against plant pathogenic fungus Cladosporium cucumerinum at 1 xcexcg in TLC bioassay (Ohtani Kazuhiro et al., Phytochem, 1993, 33:83).
The main object of the present invention is to provide a novel compound oleanane triterpenoid oligoglycoside of formula 1 of the accompanying drawing.
Another object of the present invention is to provide a process for the isolation of the said compound from a mangrove plant Aegiceras corniculatum (Blanco), belonging to the family Myrsinaceae.
Still another object of the present invention is to study the anti-fungicidal property of the compound.
Yet another object of the present invention is to provide a composition containing the compound useful as fungicidal and food preservative.
The invention relates to a novel compound oleanane triterpenoid oligoglycoside of formula 1 of the accompanying drawing (trivially known as corniculatonin). The invention also provides a process for the preparation of the compound from a mangrove plant Aegiceras corniculatum (Blanco) belonging to family Myrsinaceae. The invention further provides a composition containing the compound useful as fungicidal and food preservative and a process for preparing the same.
According to the invention there is provided, a novel compound oleanane triterpenoid oligoglycoside (trivially called corniculatonin) of formula 1 of the accompanying drawing, which is hereinafter described in detail. More particularly, this invention relates to the discovery of antifungal properties for a novel composition of corniculatonin, which has therefore been isolated from a mangrove Aegiceras corniculatum (Blanco). These properties include inhibition of the growth of the opportunistic fungi Candida sp. and Cryptococcus neoformans in the disc diffusion assays. In this assay, the compound of formula I of the accompanying drawings was fungicidal for Candida sp., Cryptococcus neoformans, Microsporum gypseum, Trichophyton mentagrophytes, T. tonsurans, T. violaceum and the food spoilage fungi Aspergillus fresenii. Except the two opportunistic fungi, other fungi tested are resistant to nystatin.
In fact, the invention relates to a novel antifungal composition with a novel compound oleanane triterpenoid oligoglycoside, named as corniculatonin of formula 1 of the accompanying drawing, derived from a mangrove plant, Aegiceras corniculatum (Blanco), belonging to family Myrsinaceae. The compound is endowed with a marked wide range of antimycotic activity. The activity was demonstrated by disc diffusion method and has been evaluated in vitro as MIC (minimum inhibition concentration) against the fungal strains of Candida sp., Cryptococcus neoformans, Trichophyton mentagrophytes, T. tonsurans, T. violaceum, Microsporum gypseum and against the mould Aspergillus fresenii and A. niger. The compound is effective in infections caused by Candida sp., Cryptococcus neoformans, Trichophyton mentagrophytes. T. tonsuras, T. violaceum and Microsporum gypseum and against the food spoilage mould Aspergillus fresenii. Amphotericin B and nystatin have been taken as reference compounds. The compound of formula 1 of the accompanying drawings is comparable in activity against yeast, Cryptococcus neoformans and Candida sp. one of the strains on which the compound acts are resistant to nystatin, which makes the compound a better candidate than the reference compound, nystatin. All the strains of fungi tested are sensitive to amphotericin B as well as the novel compound but the novel compound of formula 1 of the accompanying drawings is slightly less potent than the antibiotic.
In an embodiment of the invention, the present invention provides a novel compound oleanane triterpenoid oligoglycoside of the formula 1 of the accompanying drawing (trivially called corniculatonin) wherein R1 is selected from the group comprising of xe2x88x92OH, xe2x88x92OAc and xe2x95x90O; and R2 is a carbohydrate moiety.
In another embodiment of the present invention, the R1 can have xcex1 and xcex2 configuration.
In still another embodiment of the present invention, the carbohydrate is a polysaccharide with monosaccharide having D or L rotation and intersugar linkages have xcex1 and xcex2 configuration.
In yet another embodiment of the present invention, the carbohydrate is linked to an aglycone at position 3 and has xcex2 configuration.
In one more embodiment of the present invention, the carbohydrate moiety comprises of glucose, rhamnose and glucorinic acid.
In one another preferred embodiment of the present invention, C13-C28 oxydo ring has xcex2 configuration.
In an preferred embodiment of the present invention, the novel R2 is 
In an embodiment of the present invention, the compound inhibits the growth of the opportunistic fungi candida sp. and Cryptococcus neoformans in disc diffusion assays.
In another embodiment of the present invention, the compound inhibits growth of Microsporum gypseum, Trichophyton mentagrophytes, T. tonsurans and T. violaceum. 
In still another embodiment of the present invention, the compound inhibits growth of Aspergillus fresenii and A. niger. 
In yet another embodiment of the present invention, the compound is used for the treatment of candidiasis, cryptococcosis, epidermal infections and systemic infections.
In one more embodiment of the present invention, the compound is used as a food preservative.
The present invention also provides a process for the isolation of the compound oleanane triterpenoid oligoglycoside of formula 1 of the accompanying drawing, said process comprising contacting the plant parts with a organic solvent to obtain an extract; fractionating the extract by known methods; and isolating the compound from the fractionated extract by known methods.
In an preferred embodiment of the present invention, the process for the isolation of the compound comprises:
1. collecting and drying the plant parts of Aegiceras corniculatum; 
2. immersing the plant parts in an organic polar solvent for a week;
3. filtering the extract to obtain a filtrate;
4. concentrating the filtrate by known methods;
5. treating the concentrate with polar solvents in increasing order of polarity to obtain different layers; and
6. subjecting the thus obtained layers to fractionation to get the compound.
In an embodiment of the present invention, the organic solvent is selected from the group comprising of ethyl acetate, methanol, heptane, hexane, isooctane, acetone, benzene, toluene, diethyl ether, methylene chloride, chloroform, butanol, ethanol, isopropanol, 1,2 dichloroethane and mixtures thereof.
In another embodiment of the present invention, the solvents are selected from the group comprising of ethyl acetate and methanol.
In still another embodiment of the present invention, the plant parts are selected from the group comprising of leaves, stems, branches and roots.
In yet another embodiment of the present invention, the fractionation is performed by column chromatography.
In one more embodiment of the present invention, the column chromatography is performed by XAD-2, Sephadex LH20, and silica gel.
In one another embodiment of the present invention, the solvents used for eluting are selected from the group comprising of heptane, methanol, dichloromethane, ethyl acetate, hexane, isooctane, chloroform, 1,2 dichloroethane, benzene, toluene, isopropanol, butanol, water, ethanol, diethyl ether and mixtures thereof. Particularly preferred eluents are chloroform, methanol and mixtures thereof.
The present invention further provides a composition having an effective amount of the compound oleanane triterpenoid oligoglycoside of formula 1 of the accompanying drawing optionally along with a pharmaceutically acceptable carrier.
In an embodiment of the present invention, the composition inhibits the growth of the opportunistic fungi candida sp. and Cryptococcus neoformans in disc diffusion assays.
In another embodiment of the present invention, the composition inhibits growth of Microsporum gypseum, Trichophyton mentagrophytes, T. tonsurans and T. violaceum. 
In still another embodiment of the present invention, the composition inhibits growth of Aspergillus fresenii and A. niger. 
In yet another embodiment of the present invention, the composition is used for the treatment of candidiasis, cryptococcosis, epidermal infections and systemic infections.
In one more embodiment of the present invention, the composition is used as a food preservative.
Further more, the present invention provides a method of treating fungal infections in mammals by administering an effective amount of compound of formula 1 of the accompanying drawing or the extract obtained from the mangrove plant Aegiceras corniculatu. 
In an embodiment of the present invention, 6.25 xcexcg to 50 xcexcg/ml of the compound or the extract is administered to the subject.
In another embodiment of the present invention, the subject is a human being.